The toxicity test using D. magna is well documented in the OECD guidelines 202 and it is determined by immobilization at 24 and 48 hours after exposure to nanoparticles. In most cases, it is difficult to determine accurate toxic effects based on the method because the data varies depending on the different groups. Considering this limitation, we tried to test the acute toxicity on D. magna using influential indices including immobilization, heart rate, swimming performance and ROS level.
First, we determined the LC50 values for each material as the first step in the exposure test using organisms. The LC50 value of TiO2 in D. magna after 48 hours was determined to be more than 100 mg/ml and the values of AgNPs and Ag+ were 0.0134 and 0.0016 µg/ml, respectively. Ribeiro et al. (2014) determined the LC50 of AgNPs to be 11.02 µg/L after 48 hours of exposure, and 1.05 µg/L for silver nitrate (AgNO3) using D. magna35. Shen et al. (2015) also calculated LC50 values of 0.58–2.51 µg/L for AgNO3, which were lower than our results36. The results occur depending on the strain and water physicochemical parameters, which means that even if we measured the LC50 considering experimental conditions, the outcomes might be different between laboratories.
The change in heart rate as an index of toxic effects on D. magna is widely used in acute toxicity tests. The heart rate was measured repeatedly using more than 10 neonates exposed to the materials and a cross check for all measurements was also performed to minimize bias by observers. The results showed that the heart rate decreased with increasing concentration for all substances. It was possible to obtain a similar pattern even for 3-hour exposure with the OECD guidelines (48-hour exposure)37. Thus, we confirmed that an exposure duration of 3 hours was sufficient to test acute toxicity. In addition, TiO2 NPs considered to be non-toxic, also reduced the heart rate by increasing the exposure level. This showed that TiO2 NPs affect the heart rate of D. magna, similar to other toxic NPs (e.g., AgNPs), but it is limited to conclude that TiO2 NPs influence the immobilization (behavioral performances).
Locomotion-based behavior is a highly sensitive index for identifying toxic effects of chemicals28, 38. Thus, immobilization was characterized by measuring swimming speed (video tracking) in addition to heart rate counting. In particular, the behavioral change was measured at each exposure duration (3, 6, 12, 24, 48 hours), thus we could identify the temporal effects in detail compared to most previous studies that examined tests after 48 hours of exposure according to the OECD guidelines 202 (Fig. A1-A3 in the Supplementary Information).
To date, information on not only the temporal variations in immobilization but also the relationship between heart rate and behavioral responses to the toxic effects of nanoparticles remains limited. For example, Lovern et al. (2007) examined behavioural and physiological changes in D. magna using TiO2 NPs, fullerenes and fullerene derivatives31. The authors found that an increased exposure level influenced the low heart rate and movement. Additionally, the effect was not statistically significant for D. manga exposed to TiO2 NPs, but the temporal variation was not characterized. In our study, the heart rate increased after 48 hours of exposure compared with that after 3 hours of exposure, while it decreased in proportion to the exposure level (Fig. 2). The swimming speed at an earlier exposure duration (3 hours) was low for all materials and increased as time elapsed (Fig. 4). Therefore, it can be inferred that the exposure duration has more influences on the cardiac and behavioral effects at earlier periods, and D. magna are more active after a certain stabilization period.
The ROS level was additionally measured to support the outcomes in this study. The ROS levels of the target materials increased with the exposure level, but they gradually decreased from a certain level after 48 hours of exposure. The relative intensities of ROS were observed with inverted U-shaped patterns after 48 hours of exposure. This can be interpreted as the ROS levels recovered by enhanced antioxidative responses at high concentrations39, 40 and the overall fluorescence is enhanced by ROS production from the nanoparticles. The excessive ROS generation can cause oxidative stress41. Previous studies have also reported that oxidative stress can cause toxic effects on heart rate, swimming speed, and reproduction in D. magna42–44. In this study, it was observed that the nanoparticles enhanced the ROS levels compared to the control group, which is also consistent with previous studies13, 45.
There have been significant differences between the results on immobilization, cardiac effects and behavioral changes in previous studies. Therefore, a comprehensive test including these indices would be necessary to improve the quality of the results on acute toxicity. Furthermore, the effects of nanomaterial mixtures on aquatic toxicity need to be explored in terms of invertebrate heart physiology and swimming patterns46, 47.